Early detection of exon 1 huntingtin aggregation in zQ175 brains by molecular and histological approaches

Abstract Huntingtin-lowering approaches that target huntingtin expression are a major focus for therapeutic intervention for Huntington’s disease. When the cytosine, adenine and guanine repeat is expanded, the huntingtin pre-mRNA is alternatively processed to generate the full-length huntingtin and HTT1a transcripts. HTT1a encodes the aggregation-prone and highly pathogenic exon 1 huntingtin protein. In evaluating huntingtin-lowering approaches, understanding how the targeting strategy modulates levels of both transcripts and the huntingtin protein isoforms that they encode will be essential. Given the aggregation-propensity of exon 1 huntingtin, the impact of a given strategy on the levels and subcellular location of aggregated huntingtin will need to be determined. We have developed and applied sensitive molecular approaches to monitor the levels of aggregated and soluble huntingtin isoforms in tissue lysates. We have used these, in combination with immunohistochemistry, to map the appearance and accumulation of aggregated huntingtin throughout the CNS of zQ175 mice, a model of Huntington’s disease frequently chosen for preclinical studies. Aggregation analyses were performed on tissues from zQ175 and wild-type mice at monthly intervals from 1 to 6 months of age. We developed three homogeneous time-resolved fluorescence assays to track the accumulation of aggregated huntingtin and showed that two of these were specific for the exon 1 huntingtin protein. Collectively, the homogeneous time-resolved fluorescence assays detected huntingtin aggregation in the 10 zQ175 CNS regions by 1–2 months of age. Immunohistochemistry with the polyclonal S830 anti-huntingtin antibody showed that nuclear huntingtin aggregation, in the form of a diffuse nuclear immunostain, could be visualized in the striatum, hippocampal CA1 region and layer IV of the somatosensory cortex by 2 months. That this diffuse nuclear immunostain represented aggregated huntingtin was confirmed by immunohistochemistry with a polyglutamine-specific antibody, which required formic acid antigen retrieval to expose its epitope. By 6 months of age, nuclear and cytoplasmic inclusions were widely distributed throughout the brain. Homogeneous time-resolved fluorescence analysis showed that the comparative levels of soluble exon 1 huntingtin between CNS regions correlated with those for huntingtin aggregation. We found that soluble exon 1 huntingtin levels decreased over the 6-month period, whilst those of soluble full-length mutant huntingtin remained unchanged, data that were confirmed for the cortex by immunoprecipitation and western blotting. These data support the hypothesis that exon 1 huntingtin initiates the aggregation process in knock-in mouse models and pave the way for a detailed analysis of huntingtin aggregation in response to huntingtin-lowering treatments.


Supplementary
Optimization of antibody concentrations was performed on cortical lysates from 12-month-old zQ175 mice for assays that detect aggregated HTT: 4C9-S830, 4C9-MW8 and MW8-2B7 and from 2-month-old zQ175 mice for the soluble HTTexon1 assay. The donor antibody concentration was kept constant at 1 ng/well, whilst the acceptor antibody concentration was titrated from 1 ng/well to 40 ng/well. The maximum concentration prior to saturation was chosen as optimal (arrow). (B) Two-fold serial dilutions of 1.25 -10 μL cortical lysate from zQ175 mice was performed by diluting with age-matched wild-type lysate. The titration curve for the optimal antibody concentration is indicated (arrow). The change in fluorescent signal is denoted as ΔF%. N = 3. Optimization of antibody concentrations was performed on cortical lysates from 6-month-old wild-type and zQ175 mice for the assays that detect soluble wild-type endogenous mouse HTT: MAB2166-CHDI-1414 and total soluble full-length HTT (mutant and wild-type): D7F7-MAB5490. The donor antibody concentration was kept constant at 1 ng/well, whilst the acceptor antibody concentration was titrated from 1 ng/well to 40 ng/well. The maximum concentration prior to saturation was chosen as optimal (arrow). (B) Two-fold serial dilutions of 1.25 -10 μL cortical lysate from zQ175 mice was performed by diluting with lysis buffer. The titration curve for the optimal antibody concentration is indicated (arrow). The change in fluorescent signal is denoted as ΔF%. N = 3. WT = wild type.

Supplementary Figure 3
Supplementary Figure 3. Comparative increase in aggregated HTT in ten CNS regions from zQ175 mice aged from 1 -6 months. Aggregated HTT, as detected by the 4C9-S830 assay, increased in all brain regions from 1 -6 months of age. The presence of aggregated HTT could be first detected at 1 month in all regions except for colliculus, brain stem and spinal cord. The greatest level of aggregated HTT was in the striatum followed by the cortex, with the lowest levels in the thalamus and brain stem. In four of the brain regions, aggregated HTT levels continued to increase up to 6 months of age, whereas in the others, the levels had started to plateau between 5 and 6 months. N = 6 / genotype / age. Error bars are mean ± SEM. The test statistic, degrees of freedom and p values for the two-way ANOVA are provided in Supplementary Table 3. Statistical differences are indicated when there is a difference from one month to the next and to indicate the age at which aggregated HTT could first be detected *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. WT = wild type, AU = arbitrary units.

Supplementary Figure 4
Supplementary Figure 4. Comparative increase in aggregated HTTexon1 in ten CNS regions from zQ175 mice aged from 1 -6 months. Aggregated HTTexon1, as detected by the 4C9-MW8 assay increased in all brain regions from 1 -6 months of age. In all cases, the presence of aggregated HTTexon1 could be first detected above background at 2 months. The greatest level of aggregated HTTexon1 was in the striatum followed by the cortex, with the lowest in the brain stem and spinal cord. In seven of the brain regions, the levels had started to plateau between 5 and 6 months, although aggregated HTT continued to increase up to 6 months of age in the hippocampus, cerebellum and thalamus. N = 6 / genotype / age. Error bars are mean ± SEM. The test statistic, degrees of freedom and p values for the two-way ANOVA are provided in Supplementary Table 4. Statistical differences are indicated when there is a difference from one month to the next and to indicate the age at which aggregated HTTexon1 could first be detected *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. WT = wild type, AU = arbitrary units.

Supplementary Figure 5
Supplementary Figure 5. Comparative increase in aggregated HTTexon1 in ten CNS regions from zQ175 mice aged from 1 -6 months. Aggregated HTTexon1, as detected by the MW8-2B7 assay increased in all brain regions from 1 -6 months of age except brain stem and spinal cord. The presence of aggregated HTTexon1 could be detected above background in the striatum and cortex at 1 month. The greatest level of aggregated HTTexon1 was in the striatum followed by the olfactory bulb, with none detected in the hypothalamus, thalamus, brain stem and spinal cord. N = 6 / genotype / age. Error bars are mean ± SEM. The test statistic, degrees of freedom and p values for the two-way ANOVA are provided in Supplementary Table 5. Statistical differences are indicated when there is a difference from one month to the next and to indicate the age at which aggregated HTTexon1 could first be detected *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. WT = wild type, AU = arbitrary units. Coronal sections from zQ175 and wild-type mice at 6 months of age were immunoprobed with EPR5526 (1 -17 amino acids) and MAB5490 (115 -129), MAB2166 (443 -457) and D7F7 (around Pro1220). In contrast to S830, there was no staining above wild-type levels with any of these antibodies in the cortex, striatum or hippocampus. N = 3. Scale bar = 20 µm.

Supplementary Figure 9
Supplementary Figure 9. The 2B7 epitope was not exposed by antigen retrieval with formic acid. Immunohistochemistry with the 2B7 and 4H7H7 antibodies to striatal sections from zQ175 and wild-type mice at 6 months of age that had, or had not, been pretreated with formic acid. Neither 4H7H7 nor 2B7 detected HTT aggregates on zQ175 sections without formic acid treatment. Antigen retrieval with formic acid exposed the polyglutamine 4H7H7 epitope, but not that for 2B7. Aggregates were only detected with 4H7H7. No staining was observed in the wild-type controls. Scale bar = 5 µm. WT = wild type, FA = formic acid.
Supplementary Figure 10. Comparative decrease in soluble HTTexon1 levels in nine CNS regions from zQ175 mice aged from 1 -6 months. The level of soluble HTTexon1 as detected by the 2B7-MW8 HTRF assay decreased in all brain regions from 1 -6 months of age except for cerebellum. The brain regions with the highest levels of HTTexon1 were broadly comparable to those with the highest levels of HTT aggregation, as measured by HTRF. N = 6 / genotype / age. Error bars are mean ± SEM. The test statistic, degrees of freedom and p values for the two-way ANOVA are provided in Supplementary Table 8. Statistical differences are indicated when there is a difference from one month to the next and to indicate the age at which the level of soluble HTTexon1 were first found to decrease *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. WT = wild type, AU = arbitrary units. Figure 11. Comparative level of endogenous mouse HTT in nine CNS regions from zQ175 and wild-type mice aged from 1 -6 months. Endogenous mouse HTT was detected using the MAB2166-CHDI-1414 assay. The levels were higher in wild-type mice that have two copies of endogenous mouse HTT, compared to zQ175 mice that have one copy. Endogenous mouse HTT levels did not change in zQ175 mice over the 6-month period in any brain region. There was some variability in HTT levels in five brain regions in wild-type mice, but overall, the levels were stable. N = 6 / genotype / age. Error bars are mean ± SEM. The test statistic, degrees of freedom and p values for the two-way ANOVA are provided in Supplementary Table 9. Statistical differences indicate the age at which levels in endogenous mouse HTT had first changed *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. WT = wild type, AU = arbitrary units.

Supplementary Figure 12
Supplementary Figure 12. Comparative level of total full-length HTT in nine CNS regions from zQ175 and wild-type mice aged from 1 -6 months. Total full-length HTT (mutant and wild-type) was detected using the D7F7-MAB5490 assay. The levels of total full-length HTT were comparable between wild-type and zQ175 mice. There was no consistent change in total HTT levels in zQ175 mice between brain regions. In the cortex, colliculus and spinal cord, there was a comparable decrease in total HTT levels in wild-type and zQ175 mice over the six-month period, suggesting that this is not due to the influence of the HTT mutation. Overall, the levels of total HTT were stable. N = 6 / genotype / age. Error bars are mean ± SEM. The test statistic, degrees of freedom and p values for the two-way ANOVA are provided in Supplementary  Table 10. Statistical differences indicate the age at which levels in total full-length HTT had first be found to change *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. WT = wild type, AU = arbitrary units.
Supplementary Figure 13. Full-sized loading control blot. The full-sized loading control blot immunoprobed with HDAC4 for the western blot presented in Fig. 9C. The molecular weight markers are in kDa. WT = wild type.
Supplementary Table 3. The test statistic, degrees of freedom and p values for the two-way ANOVA of data presented in Fig. 2A and Supplementary Fig. 3.  Fig. 2C and Supplementary Fig. 5.